Method for preparing antibody monolayers which have controlled orientation using peptide hybrid

ABSTRACT

The present invention relates to a method for preparing an protein monolayer using a peptide hybrid for protein immobilization, more precisely a peptide hybrid for protein immobilization which has improved solubility by introducing a PEG linker and a proper reaction group to the oligopeptide having specific affinity to selected types of proteins and is designed to provide enough space between solid substrates and proteins immobilized, whereby various solid substrates treated by the hybrid catch specific proteins effectively on. The peptide hybrid for protein immobilization of the present invention facilitates the control of orientation of an antibody on various solid surfaces and immobilization of various antibodies of different origins or having different isotypes with different affinity. Therefore, the surface treatment technique using the peptide hybrid of the invention can be effectively used for the production of various immunosensors and immune chips.

TECHNICAL FIELD

The present invention relates to a method for preparing an proteinmonolayer using a peptide hybrid for protein immobilization, moreprecisely a peptide hybrid for protein immobilization which has improvedsolubility by introducing a PEG linker and a proper reaction group tothe oligopeptide having specific affinity to selected types of proteinsand is designed to provide enough space between solid substrates andproteins immobilized, whereby various solid substrates treated by thehybrid catch specific proteins effectively on.

BACKGROUND ART

Antibodies have been immobilized on the surfaces of various inorganicsolid materials for producing immunosensor, protein chip, diagnostickit, etc. The technique to regulate orientation in order to expose theantigen binding site of an antibody on the surface without damaging theactivity and binding capacity peculiar to the antibody and thus toimmobilize the antibody on the various solid substrates as a form ofmonolayer is very important since it is directly involved in thedetecting sensitivity of a sensor or a chip.

The conventional methods to immobilize an antibody on the surface of asolid substrate rely on physical adsorption or covalent bond formationof a protein. However, the immobilization of an antibody by theconventional methods has disadvantages of protein denaturation, randomorientation, decreased binding capacity of the immobilized antibody tothe target antigen resulting from random chemical modifications.

To overcome the above problems, antibody immobilization techniques havebeen developed by using microorganism-originated antibody bindingproteins (protein A, protein G, protein A/G or protein L) bindingspecifically to the corresponding antibody. These proteins bind stronglyto a specific region of an antibody that is not involved in theantigen-antibody reaction, immobilizing the antibody on a solidsubstrate to allow the approach of an antigen. The interaction betweenthe above proteins and antibodies does not require any chemicalmodification process, suggesting that the unique antibody functions arenot damaged. However, it is very difficult to regulate orientation ofthe proteins during immobilization on a solid substrate, and as aresult, the antibody immobilization efficiency might be reduced. Recentstudies have been focused on the modification of an antibody bindingprotein by genetic engineering and chemical approaches to overcome theabove problem.

The antibody immobilization using an antibody binding protein has manyadvantages, compared with the method based on physical adsorption, butstill has a problem of protein denaturation by many environmentalfactors including physical and chemical factors, suggesting thatlong-term storage is difficult. It is also almost impossible to treat aspecific target region only in case a chemical modification is necessaryin a specific region of a protein. It is an urgent request, to overcomesuch problems, to develop a novel method for antibody immobilizationusing a low molecular weight material having high stability andfacilitating immobilization on a solid substrate. Antibodyimmobilization methods have been developed using a dendrimer, iron ionor calixcrown derivatives, but these methods do not have the control oforientation and selectivity, suggesting that the antibody does not haveprotein specificity, that is it can be bound to almost every proteinswith similar binding capacity which is though not very strong.

To screen a novel low molecular substance for protein immobilization inorder to overcome the disadvantages of the conventional antibody bindingprotein or low molecular substance for antibody immobilization, thepresent inventors carried out wide document analysis to investigatewhich low molecular substance could be used as an adsorption materialfor antibody separation and as a therapeutic agent based on antibodybinding. The present inventors selected three kinds of peptides bindingselectively to IgG as candidates for the low molecular substance of theinvention (DeLano W L et al., Science 287:1279-1283, 2000; Yang H etal., J Peptide Res 66(Suppl. 1):120-137, 2006; Fassina G et al., J MolRecognit 11:128-133, 1998), and prepared peptide hybrids for proteinimmobilization by modifying chemical structures of the peptides, andfinally completed this invention by confirming that the peptide hybridsfor protein immobilization had binding capacity about antibody andantigen on the solid substrate for the immunosensor and antibody chip.

DISCLOSURE Technical Problem

It is an object of the present invention to provide a bio-chip or animmunosensor with satisfactory surface regularity by inhibitingnon-specific binding reaction on the solid substrate during antibodyimmobilization and by regulating antibody orientation.

Technical Solution

To achieve the above object, the present invention provides a peptidehybrid for protein immobilization composed of an oligopeptide comprising7-17 amino acids having a specific protein specific affinity and PEGlinked to the oligopeptide by covalent bond.

The present invention also provides a method for preparing a proteinmonolayer comprising the following steps:

1) activating the carboxyl group included in a solid substrate;

2) surface-treating the solid substrate having the activated carboxylgroup by adhering the peptide hybrid for protein immobilization of thepresent invention onto the surface; and,

3) binding a protein onto the surface-treated solid substrate of step2),

and a substrate for protein immobilization surface-treated with thepeptide hybrid for protein immobilization of the invention prepared bythe same.

The present invention further provides an immunosensor having anantibody immobilized via the peptide hybrid on the solid substratesurface-treated with the peptide hybrid for protein immobilization ofthe invention.

The present invention also provides a detection method of an antigencomprising the step of detecting the antigen-antibody reaction afteradding the sample antigen to the antibody immobilized on theimmunosensor.

In addition, the present invention provides a detection method of anantibody in the sample, comprising the following steps:

1) adding an antigen binding specifically to the antibody immobilized onthe immunosensor and washing thereof;

2) adding a sample to the washed immunosensor of step 1) and washingthereof; and,

3) detecting the antigen-antibody reaction between the added antigen andthe immobilized antibody.

Advantageous Effect

The peptide hybrid for protein immobilization of the present inventionprovides orientation to an antibody on various types of solid substratesurfaces and facilitates the immobilization of antibodies with differentaffinities according to their origins or subtypes. Thus, the surfacetreatment technique using the peptide hybrid of the invention can beeffectively used for the production of various immunosensors and immunechips.

DESCRIPTION OF DRAWINGS

The application of the preferred embodiments of the present invention isbest understood with reference to the accompanying drawings, wherein:

FIG. 1 is a schematic diagram illustrating the principle of antibodyimmobilization on a solid substrate by the peptide hybrid for proteinimmobilization of the present invention.

FIG. 2 is a set of graphs illustrating the binding capacities of human,rabbit, mouse and goat antibodies and BSA to the dextran CM-5 Au sensorchip surface-treated with the peptide hybrid for protein immobilizationof the invention measured by surface plasmon resonance:

a: results on the chip surface-treated with the hybrid represented byformula 1; and,

b: results on the chip surface-treated with the hybrid represented bythe formula 2.

FIG. 3 is a set of graphs illustrating the binding capacities of humanand mouse antibodies to the dextran CM-5 Au sensor chip surface-treatedwith the peptide hybrid for protein immobilization of 1a of theinvention measured by surface plasmon resonance:

a: binding capacities of human antibodies HIgG1, HIgG2 and HIgG3measured by surface plasmon resonance; and,

b: binding capacities of mouse antibodies MIgGA, MIgG1, MIgG2 and MIgG3measured by surface plasmon resonance.

FIG. 4 is a graph illustrating the binding affinity between the peptidehybrid of 1a and human, rabbit, mouse and goat antibodies:

a: binding capacity of human IgG1 (anti CRP) antibody (100 nM, 250 nM,500 nM and 1,000 nM) to the dextran CM-5 Au sensor chip surface-treatedwith the peptide hybrid for protein immobilization of 1a of theinvention measured by surface plasmon resonance; and

b: binding affinity between the peptide hybrid for proteinimmobilization of 1a of the invention and HIgG1, rabbit IgG and MIgG3antibodies.

FIG. 5 is a graph illustrating the antigen binding capacity of theantibody immobilized to the dextran CM-5 Au sensor chip surface-treatedwith the peptide hybrid for protein immobilization of 1a of theinvention.

FIG. 6 is a graph illustrating the comparison of antigen-antibodybinding capacity between the anti-CRP antibody chip (peptidehybrid-anti-CRP) immobilized on the chip surface-treated with thepeptide hybrid of 1a of the invention and the anti-CRP antibody chip(chemical anti-CRP) immobilized on the surface of the chip by chemicalmethod.

FIG. 7 is a photograph illustrating the binding capacities of human,mouse, goat and rabbit antibodies to the magnetic micro beadssurface-treated with the peptide hybrid of 1a of the invention measuredby PAGE.

FIG. 8 is a set of photographs illustrating the binding capacities ofhuman, mouse, goat and rabbit antibodies to the glass platesurface-treated with the peptide hybrid of 1a of the invention measuredby PAGE.

MODE FOR INVENTION

Hereinafter, the present invention is described in detail.

The present invention provides a peptide hybrid for proteinimmobilization composed of an oligopeptide comprising 7-17 amino acidshaving a specific protein specific affinity and PEG linked to theoligopeptide by covalent bond.

The protein herein can be any protein accepted by those in the art, inparticular a protein having biological activity selected from the groupconsisting of proteins for medicinal purpose, research and industry, forexample, antigen, antibody, cell receptor, enzyme, structural protein,serum, and cellular protein, and more preferably an antibody. If theprotein is an antibody, the oligopeptide has an affinity to Fc of theantibody.

The peptide hybrid for protein immobilization of the invention ispreferably composed of 7-17 amino acids for the control of orientationduring the protein immobilization on various solid surfaces, and morepreferably composed of 13-17 amino acids. The peptide can contain aminoacid sequences represented by SEQ. ID. NO: 1-NO: 5 included in thepartial structures of the peptide hybrids represented by formulas 1-3and formula 4 (SEQ. ID. NO: 1: DDDC*AWHLGELVWC*T; SEQ. ID. NO: 2:DEDC*AWHLGELVWC*T; SEQ. ID. NO: 3: EEDC*AWHLGELVWC*T; SEQ. ID. NO: 4:EDDC*AWHLGELVWC*T; formula 4: (RTY)₄K₂KG; SEQ. ID. NO 5: GHWRGWVS, C*:disulfide bond) (see Table 1). These peptides are known to bind to Fcsite of human immunoglobulin G (DeLano W L et al., Science287:1279-1283, 2000; Yang H et al., J Peptide Res 66(Suppl. 1):120-137,2006; Fassina G et al., J Mol Recognit 11:128-133, 1998).

The preferable molecular weight of the PEG (poly ethylene glycol) is60-3000. The peptide hybrid designed to have the PEG can have improvedsolubility in a buffer solution and can provide enough space necessaryfor the effective antibody binding in between the solid substrate andthe peptide having the amino acid sequence selected from the groupconsisting of sequences represented by SEQ. ID. NO: 1-NO: 5 andrepresented by formula 4 (see FIG. 1).

The peptide hybrid for protein immobilization of the present inventionallows various chemical modifications and is preferably one of thepeptide hybrids represented by formulas 1-3 containing the peptidehaving the amino acid sequence selected from the group consisting ofsequences represented by SEQ. ID. NO: 1-NO: 5 and formula 4 shown inTable 1, and is more preferably the peptide hybrid represented byformula 1a comprising the peptide having the amino acid sequencerepresented by SEQ. ID. NO: 4.

Formula 1a H₂N (CH₂CH₂O)₂CH₂CH₂COEDDC*AWHLGELVWC*T-CONH₂ (C*: disulfidebond; EDDC*AWHLGELVWC*T: SEQ. ID. NO: 4)

The chemical modification herein can be achieved by differentDiels-Alder reaction substrates including photoreactive functionalgroup, thiol specific functional group (maleimide, etc), biotin, NTA(nitrilotetra-acetic acid), IDA (iminodiacetic acid), maltose orspecific reactive functional group (diene, dienophile), etc. The peptidehybrid for protein immobilization of the invention can be furtherapplied to additional protein binding or conjugation of a specificbioactive substance through the chemical modification. This chemicalmodification also facilitates the development of an antibody therapeuticagent, precisely the modified peptide hybrid is acting as a carrier todeliver a therapeutic agent such as a specific compound, a bioactivepeptide and a protein or a radioisotope to target region such as cancercells. In a preferred embodiment of the invention, the peptide hybridfor protein immobilization of the present invention is used for antibodyimmobilization with different binding capacities according to theirorigins or subtypes, when the target protein is an antibody. The peptidehybrid represented by formula 1 can be strongly bound to human andrabbit originated antibodies (see FIG. 2 a) and the peptide hybridrepresented by formula 1a can be particularly strongly bound to humanoriginated HIgG1 (K_(d)=85 nM) and HIgG2 and rabbit originated IgG(K_(d)=305 nM) (see FIGS. 3 and 4).

The present invention also provides a method for preparing a proteinmonolayer comprising the following steps:

1) activating the carboxyl group included in a solid substrate;

2) surface-treating the solid substrate of step 1) with the peptidehybrid for protein immobilization of the invention; and,

3) binding a protein onto the surface-treated solid substrate of step2),

and a substrate for protein immobilization surface-treated with thepeptide hybrid for protein immobilization of the invention prepared bythe same.

The solid substrate above can be selected from the group consisting ofCM-5 Au sensor chip, magnetic micro beads, glass plate, gold nanoparticles, biodegradable organic polymer nano particles such as PLGA orvarious (micro) well plates. The solid substrate characteristicallycontains a carboxyl group and once this carboxyl group is activated itwill be reacted with the terminal amine group of the peptide hybrid forprotein immobilization of the invention to immobilize the hybrid. Thesurface-treatment on a solid substrate facilitates the protein bindingwith controlled orientation and increases the surface regularity of thesolid substrate. According to the preparing method of a monolayer usingthe peptide hybrid for protein immobilization of the present invention,particularly when the protein herein is an antibody, physical andchemical stability is improved, compared with the conventional methodusing an antibody binding protein (protein A, protein G, protein A/G orprotein L), various chemical modifications are allowed with extendingthe range of applications, and antibody selection and binding capacityare also increased compared with the conventional method using a lowmolecular weight compound.

The hybrid is preferably one of the peptide hybrids for proteinimmobilization represented by formulas 1-3 comprising the peptide havingthe amino acid sequence selected from the group consisting of sequencesrepresented by SEQ. ID. NO: 1-NO: 5 and formula 4 shown in Table 1, andis more preferably the peptide hybrid for protein immobilizationrepresented by formula 1a comprising the peptide having the amino acidsequence represented by SEQ. ID. NO: 4.

The antibody is human, rabbit, mouse or goat originated immunoglobulinG. If the substrate is treated with the peptide hybrid for proteinimmobilization represented by formula 1a, the antibody is preferablyhuman or rabbit originated immunoglobulin G.

The various solid substrate surfaces-treated according to the method ofthe invention can be effective in variety of antibody immobilization.Human-originated and rabbit-originated IgGs are immobilized effectivelyon all of the CM-5 Au sensor chip surface-treated with the peptidehybrid for protein immobilization of the invention represented byformula 1a (see FIGS. 2-6), magnetic micro beads (see FIG. 7) and glassplate (see FIG. 8). In the meantime, mouse-originated and goatoriginated IgGs are not successfully immobilized thereon. The method ofthe invention is also useful for the construction of a biosensor basedon micro particles and the glass plate surface-treated according to themethod of the invention has been confirmed to maintain its antibodybinding capacity after several months storage at room temperature.

The present invention further provides an immunosensor having anantibody immobilized on its solid substrate surface via the peptidehybrid of the invention.

Anti-CRP antibody was immobilized on the chip surface-treated with thepeptide hybrid for protein immobilization of 1a, and thenantigen-antibody binding was measured with spilling the antigen CRP bysurface plasmon resonance (SPR). As a result, it was confirmed that theanti-CRP antibody was bound to the antigen CRP (see FIG. 5). The surfaceof the chip treated with the peptide was so stable that the antibodybinding capacity was not reduced after washing the surface with 20 mMNaOH and reusing it for the antibody-antigen binding.

The antigen-antibody binding capacity was compared between the anti-CRPantibody chip surface-treated with the peptide hybrid for proteinimmobilization of 1a and the anti-CRP antibody chip chemically treated.As a result, the binding capacity of the chip with anti-CRP-antibodyimmobilized on its surface by using the peptide hybrid of 1a wasapproximately 1.6 fold higher than that of the chip with anti-CRPantibody immobilized by the chemical treatment (see FIG. 6).

The present invention also provides a detection method of an antigencomprising the step of detecting the antigen-antibody reaction afteradding a sample antigen to the antibody immobilized on the immunosensor.

The immunosensor can be prepared by the steps of surface-treating asolid substrate having the activated carboxyl group with the peptidehybrid and fixing a target antigen specific antibody thereon.

The antigen-antibody reaction can be detected by SPR, enzymeimmunoassay, and fluorescence assay using a fluorescent probe. Afterbinding an antigen specific antibody-coloring enzyme conjugate to anantigen and washing, a substrate reacting to the coloring enzyme wasadded, followed by measuring the color development. The coloring enzymeis selected from the group consisting of HRP (horseradish peroxidase),GUS (β-Glucuronidase), AP (alkaline phophatase), β-Gal (β-Galactosidase)and luciferase, but not always limited thereto. Or after binding anantigen-specific antibody-fluorescent probe conjugate and washing,fluorescence was measured. The fluorescent probe is selected from thegroup consisting of 6-FAM, fluorescein, Cy3, Cy5 and rhodamine, but notalways limited thereto.

The detection method of the antigen-antibody reaction depends on a solidsubstrate of the immunosensor. Particularly, if the solid substrate isCM-5 Au sensor chip, an antigen is injected on the immunosensor atregular speed, during which the antigen-antibody binding is measured bysurface plasmon resonance. In case the solid substrate is magnetic microbead, the magnetic micro bead itself is first boiled in a buffersolution and then PAGE is performed (see FIG. 7). If the solid substrateis glass plate, the peptide surface is treated with theantibody-fluorescent probe conjugate and then fluorescence from theantibody is measured (see FIG. 8).

In addition, the present invention provides a detection method of anantibody in the sample, comprising the following steps:

1) adding an antigen binding specifically to the antibody immobilized onthe immunosensor and washing thereof;

2) adding a sample to the washed immunosensor of step 1) and washingthereof; and,

3) detecting the antigen-antibody reaction between the added antigen andthe immobilized antibody.

In step 3), the antigen-antibody reaction can be detected by SPR, enzymeimmunoassay, and fluorescence assay using a fluorescent probe. In thisstep, the secondary antibody-coloring enzyme conjugate is bound to theantibody immobilized thereon, which is washed. Then, a substratereacting to the coloring enzyme is added, followed by measuring thecolor development. The coloring enzyme is selected from the groupconsisting of HRP (horseradish peroxidase), GUS (β-Glucuronidase), AP(alkaline phophatase), β-Gal (β-Galactosidase) and luciferase, but notalways limited thereto. In step 3), the reaction can also be detected bythe following steps; the secondary antibody-fluorescent probe conjugateis bound to the antibody immobilized thereon, which is washed andproceeds to the measurement of fluorescence. The fluorescent probe isselected from the group consisting of 6-FAM, fluorescein, Cy3, Cy5 andrhodamine, but not always limited thereto.

Practical and presently preferred embodiments of the present inventionare illustrative as shown in the following Examples.

However, it will be appreciated that those skilled in the art, onconsideration of this disclosure, may make modifications andimprovements within the spirit and scope of the present invention.

Example 1 Preparation of Peptide Hybrid

The present inventors prepared the peptide hybrid for proteinimmobilization from the peptide hybrids represented by formulas 1-3comprising the peptide containing the amino acid sequence selected fromthe group consisting of the sequences represented by SEQ. ID. NO: 1-NO:5 and formula 4 shown in Table 1.

The peptides having the amino acid sequence selected from the groupconsisting of the sequences represented by SEQ. ID. NO: 1-NO: 5 andformula 4 (SEQ. ID. NO: 1: DDDC*AWHLGELVWC*T; SEQ. ID. NO: 2:DEDC*AWHLGELVWC*T; SEQ. ID. NO: 3: EEDC*AWHLGELVWC*T; SEQ. ID. NO: 4:EDDC*AWHLGELVWC*T; formula 4: (RTY)₄K₂KG; SEQ. ID. NO: 5: GHWRGWVS, C*:disulfide bond) are known to be bound to Fc region of humanimmunoglobulin G (DeLano W L et al., Science 287:1279-1283, 2000; Yang Het al., J Peptide Res 66(Suppl. 1):120-137, 2006; Fassina G et al., JMol Recognit 11:128-133, 1998). To improve solubility of the hybrid inwater-based buffer and to provide enough space in between the solidsubstrate and the peptide having the amino acid sequence selected fromthe group consisting of sequences represented by SEQ. ID. NO: 1-NO: 5and formula 4 for effective antibody binding, the peptide hybrid wasdesigned to have PEG (poly ethylene glycol). The designed peptide hybridwas prepared by solid phase synthesis. The product with 97% purityconfirmed by HPLC was purchased from BioFuture (Korea) and mass analysiswas performed by MALDI-TOF to confirm the structure.

TABLE 1 Peptide hybrid for protein immobilization Amino acid No Formulasequence 1 H₂N(CH₂CH₂O)_(n)CH₂CH₂COXDC*AWH SEQ. ID. NO: 1DDDC*AWHLGELVWC*T LGELVWC*T-CONH₂ SEQ. ID. NO: 2 DEDC*AWHLGELVWC*T SEQ.ID. NO: 3 EEDC*AWHLGELVWC*T 1a H₂N(CH₂CH₂O)₂CH₂CH₂COEDDC*AW SEQ. ID. NO:4 EDDC*AWHLGELVWC*T HLGELVWC*T-CONH₂ 2 (RTY)₄K₂KGNHCH₂CH₂(OCH₂CH₂)_(n)Formula 4 (RTY)₄K₂KG OCH₂CH2₂O-Cys-NH₂ 3 H₂NGHWRGWVS- SEQ. ID. NO: 5GHWRGWVS NH(CH₂CH₂O)nCH₂CH₂CO-Cys-NH₂ C*: disulfide bond

Example 2 Preparation of Antibody Samples and Reagents

Various immunoglobulin Gs having different isotypes and origins fromhuman (HIgG1, HIgG2 and HIgG3), rabbit, mouse (MIgGA, MIgG1, MIgG2 andMIgG3) and goat, were purchased from Sigma-Aldrich (USA) along with(3-aminopropyl)trimethoxysilane (APTS), N-hydroxysuccinimide(NHS),1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC),2-(N-morpholino)ethanesulfonic acid (MES), ethanolamine and Succinicanhydride-DMF, etc used for the antibody labeling. C-reactive protein(CRP) and rabbit anti-CRP antibody were purchased from Calbiochem (USA).Cys-mono NHS ester used for the antibody labeling was purchased from GEHealthcare (Korea). CM-5 Au sensor chip for the measurement of surfaceplasmon resonance was purchased from Biacore AB (Sweden), magnetic microbeads (Dynabeads MyOne™ Carboxylic Acid) were purchased from DYNAL(USA), and the glass plate was purchased from Corning (Korea).

Example 3 Antibody Binding Capacity of the Peptide Hybrid for ProteinImmobilization Immobilized on the Dextran Chip with ControlledOrientation

<3-1> Immobilization of the Peptide Hybrid with Controlled Orientation

The peptide hybrid for protein immobilization prepared in Example 1 wasimmobilized on a solid substrate having a carboxyl group on its surface.

PBS containing 0.2 M EDC and 0.05 M NHS was spilled on the dextran CM-5Au sensor chip (Biacore AB, Sweden) at the speed of 7 μl/minute toactivate the carboxyl group on the surface of the sensor chip. Then, PBScontaining 100 μM of the peptide hybrid was spilled thereon at the samespeed for 30 minutes. The peptide hybrid was bound to the surface viathe reaction of the terminal amine group on the sensor chip activated asNHS ester form. The surface area remaining non-reacted with the peptidehybrid was inactivated by using 1 M of ethanolamine solution (pH 8.5).

<3-2> Measurement of Antibody Binding Capacity

The chip surface-treated with the peptide hybrid for proteinimmobilization prepared in Example <3-1> was bound to the antibody ofExample 2.

Antibody solutions containing different animal originated antibodies ofExample 2 or 5 μg/ml of BSA (control) in PBS were spilled on the chip ofExample <3-1> at the speed of 10 μl/minute to bind the antibody to thepeptide hybrid on the surface of the chip. The binding capacity wasmeasured by surface plasmon resonance (SPR) using Biacore 3000.

As a result, the peptide hybrid for protein immobilization representedby formula 1 was strongly attached to the different types of antibodies(FIG. 2 a), while the peptide hybrids for protein immobilizationrepresented by formulas 2-3 exhibited low reaction values of up to 200RU (FIG. 2 b).

In particular, the peptide hybrid represented by formula 1a (wherein,n=2 and x=ED) (hereafter, hybrid of 1a) was bound to the humanoriginated IgG (HIgGs 1-3) and the rabbit originated IgG to thesaturated level (12000 RU). However, the hybrid of 1a was bound to therabbit originated antibodies (mixed composition of MIgGs 1-3) and to thegoat originated antibody only at minimum level but not bound to BSA(FIG. 2 a).

The hybrid of 1a was effectively bound to the human originated HIgG1 andHIgG2, but barely bound to HIgG3 (FIG. 3). The hybrid was bound to themouse originated antibody MIgG3 to some degree but hardly bound toMIgGA, MIgG1 and MIgG2 (FIG. 3 b).

<3-3> Binding Affinity of the Hybrid of 1a

Binding affinity of the hybrid of 1a confirmed to have antibody bindingcapacity in Examples <3-1> and <3-2> to the antibody was measured.

The antibody solutions containing HIgG1, rabbit IgG, MIgG3 antibodies atthe concentrations of 100 nM, 250 nM, 500 nM and 1,000 nM were spilledon the chip surface-treated with the hybrid of 1a of Example <3-1> atthe speed of 10 μl/minute to induce the binding reaction and the degreeof binding was measured by surface plasmon resonance (SPR) using Biacore3000.

As a result, SPR sensor gram values represented in dotted line wereobtained. The values were analyzed by nonlinear regression usingBIAevaluation software equipped in Biacore 3000 to draw a theoreticalline graph (FIG. 4 a). From the theoretical graph, kinetic coefficientsshown in FIG. 4 b were obtained and the overlapping of each dotted lineon the line indicates the calculation was correct.

It was confirmed that the hybrid of 1a was strongly bound to the humanoriginated HIgG1 (K_(d)=85 nM) but it had low binding affinity to therabbit originated IgG (K_(d)=305 nM) (FIG. 4 b). Therefore, the hybridof 1a was very useful for the antibody immobilization on the surface ofa chip under the control of binding degree of different antibodieshaving different origins or different isotypes.

Example 4 Antigen-Binding Capacity of the Antibody Immobilized on theChip by the Peptide Hybrid for Protein Immobilization of 1a

The antigen-binding capacity of the antibody immobilized by the hybridof 1a was measured.

<4-1> Antigen-Antibody Binding

PBS containing rabbit anti-CRP antibody at the concentration of 5 μg/mlwas spilled on the chip surface-treated with the peptide hybrid of 1aprepared in Example <3-1> at the speed of 10 μl/minute for antibodyimmobilization. Then, PBS containing CRP at the same concentration asthe above was spilled thereon, followed by measuring theantigen-antibody binding by surface plasmon resonance (SPR) usingBiacore 3000. The surface of the chip was recovered by 20 mM NaOHsolution for the repeat of the experiment to measure theantigen-antibody binding.

As a result, anti-CRP antibody was very well bound to the correspondingantigen CRP (FIG. 5). Besides, the surface treated with the peptide wasso stable even after washing with 20 mM NaOH solution that it could berecycled for further antigen-antibody binding reaction without reducingthe antibody binding capacity.

<4-2> Antigen-Antibody Binding Under the Controlled Orientation

A buffer containing 0.2 M EDC and 0.05 M NHS was spilled on the dextranCM-5 Au sensor chip (BIacore AB, Sweden) at the speed of 7 μl/minute toactivate carboxyl group on the surface of the sensor chip. Then, PBScontaining 5 μg/ml of anti-CRP antibody was spilled thereon for 30minutes at the same speed. The antibody was bound via the reaction ofthe terminal amine group with the carboxyl group on the sensor chipactivated as NHS ester form. The surface not reacted with the antibodywas inactivated by using 1 M ethanolamine solution (pH 8.5).

PBS containing 5 μg/ml of CRP was spilled on the anti-CRP antibody chipwith the chemical immobilization and the anti-CRP antibody chip with theimmobilization by the peptide hybrid of 1a respectively at the speed of10 μl/minute, followed by measuring the degree of antigen-antibodybinding by surface plasmon resonance using Biacore 3000.

As a result, the binding capacity was approximately 1.6 fold higher onthe chip having anti-CRP antibody immobilized on the surface by thepeptide hybrid of 1a than on the chip having anti-CRP antibodyimmobilized on its surface by the conventional chemical method (FIG. 6).

Example 5 Antibody Binding Capacity of the Peptide Hybrid for ProteinImmobilization of 1a Immobilized on Magnetic Micro Beads with ControlledOrientation

<5-1> Immobilization of the Peptide Hybrid of 1a with ControlledOrientation

The peptide hybrid for protein immobilization of 1a obtained in Example1 and confirmed to have antibody binding capacity in Example 3 wasimmobilized on the surface of the magnetic micro bead having a carboxylgroup on its surface.

The surface of the magnetic micro bead containing a carboxyl group wasactivated by loading 25 mM of MES (pH 6) containing NHS (0.4 M) and EDC(0.2 M). Only magnetic micro bead was recovered by using a magnet andthe activated magnetic micro bead was reacted with 0.2 mg/ml of thepeptide hybrid of 1a at room temperature for one hour. The activatedmagnetic micro bead was reacted with PBS alone for the control group.Non-reacted active ester group was inactivated by using 1 M ofethanolamine (pH 8.5). Both the magnetic micro bead with the peptidehybrid of 1a attached and the magnetic micro bead without the peptidehybrid of 1a were repeatedly washed by PBS and diluted in PBS (finalconcentration: 5 mg/ml).

<5-2> Antibody Binding Capacity

To confirm the binding of the magnetic micro bead adhered with thepeptide hybrid of 1a to the antibody, 2 mg/ml (final concentration) ofthe magnetic micro bead and 0.1 mg/ml of the antibody used in Example<3-2> were mixed to induce binding reaction. After inducing reaction forone hour at room temperature, the magnetic micro bead was dragged downto the bottom of the vessel by using a magnet and the supernatant waseliminated. This process was repeated five times to remove thenon-reacted antibodies. The magnetic micro bead reacted with theantibody was added in PAGE loading buffer containing a reducing agent(2-mercaptoethanol), which was heated to separate the antibody chainfrom the magnetic micro bead, followed by PAGE.

The antibody bound to the magnetic micro bead was quantified by using12% polyacrylamide gel containing 10% SDS. Particularly, 20 μl of PBScontaining 2% micro bead was mixed with 5 μl of sample buffer (pH 6.8;60 mM Tris-HCl, 25% glycerol, 2% SDS, 14.4 mM 2-mercaptoethanol, 0.1%bromophenol blue, H₂O), followed by heating at 90° C. for 10 minutes. 15μl of the sample was placed on each well of polyacrylamide gel. 200 V ofvoltage was applied thereto at room temperature for one hour to migratesample, followed by dipping the gel in the staining solution (0.5%coomassie blue, 45% methanol, 10% acetic acid solution) at roomtemperature for 30 minutes. Upon completion of staining, the gel wastransferred into the destaining solution (10% methanol, 10% acetic acidsolution), followed by stirring at room temperature for 3 hours. Theexcessive staining reagent was eliminated by washing.

As a result, after several times of washing with PBS, the antibodybinding was still observed on the magnetic micro bead surface-treatedwith the peptide hybrid of 1a (FIG. 7). The magnetic micro bead treatedwith the peptide hybrid of 1a was effectively bound to the humanoriginated HIgG and the rabbit originated IgG, but not to the mouse andgoat originated IgGs, which was consistent with the result of Example 3.

Example 6 Antibody Binding Capacity of the Peptide Hybrid for ProteinImmobilization of 1a Immobilized on Glass Plate with ControlledOrientation

The antibody binding capacity of the peptide hybrid for proteinimmobilization of 1a was measured.

<6-1> Immobilization of the Peptide Hybrid of 1a with ControlledOrientation

The glass plate was treated with a mixed solution of 95% sulfuric acidand 5% hydrogen peroxide (V/V 3:1) at 60° C. for 30 minutes, followed bywashing with distilled water and ethanol. The washed glass plate wassoaked in 1% APTS, followed by reaction for 4 hours at room temperatureto introduce amine group. The amine group introduced glass plate wasreacted in 1 M succinic anhydride-DMF solution at 37° C. for 4 hours togenerate carboxyl group on the glass plate. The glass plate was washedwith distilled water and ethanol and dried using nitrogen gas and thenstored in a vacuum drier. The carboxyl group introduced glass plate wastreated with a mixed solution of 0.1 M EDC and 0.025 M NHS for 15minutes to activate its surface. The activated glass plate was washedwith distilled water and dried using nitrogen gas. The peptide hybridfor protein immobilization of 1a was dissolved in PBS containing 40%glycerol at the final concentration of 0.5 mg/ml, which was dropped onthe glass plate by 1 μl per each drop, followed by reaction for 2 hours.The non-reacted active ester remaining on the glass plate wasinactivated by using 1 M of ethanolamine (pH 8.5) and washed with PBS.

<6-2> Antibody Binding Capacity

The antibody binding capacity of the glass plate surface-treated withthe hybrid of 1a was measured by using Cy3 labeled antibodies.

First, 5 μl of 3.34 mM Cy3-mono NHS ester-DMF solution was added into100 μl of each PBS respectively containing HIgG (mixture of HIgG 1-HIgG3), MIgG (mixture of MIgG 1-MIgG 3), rabbit IgG and goat IgG (0.5mg/ml), followed by reaction for 30 minutes at room temperature. Thenthe mixture proceeded to mini-gel column (PD-10 desalting column,Armersham-Biosceince, USA) to eliminate excessive fluorescent dye andfiltered protein was used for the experiment. The antibodies labeledwith Cy3 were diluted in PBS containing 0.01% Tween 20 and 0.1 μg/ml BSAat the final concentration of 1 g/ml.

The Cy3 labeled antibody solution was dropped on the glass platesurface-treated with the hybrid of 1a by 50 μl per each drop, followedby reaction for one hour at room temperature. The glass plate was washedwith PBST, PBS and distilled water in that order and dried usingnitrogen gas, followed by observation of fluorescent images on the glassplate using GenPix 4200 (Axon, USA) camera.

As a result, the human originated and rabbit originated antibodies werebound very well to the glass plate surface-treated with the hybrid of1a, while the mouse originated and goat originated antibodies were notbound well to the glass plate (FIG. 8).

Even after several months storage at room temperature, the glass platesurface-treated with the hybrid of 1a maintained its antibody bindingcapacity, suggesting that the surface treatment was very successfullyperformed providing very stable surface structure.

INDUSTRIAL APPLICABILITY

As explained hereinbefore, the peptide hybrid for protein immobilizationof the present invention provides orientation to an antibody on varioustypes of solid substrate surfaces and facilitates the immobilization ofantibodies having different affinities according to their origins orsubtypes. Thus, the surface treatment technique using the peptide hybridof the invention can be effectively used for the production of variousimmunosensors and immune chips.

Those skilled in the art will appreciate that the conceptions andspecific embodiments disclosed in the foregoing description may bereadily utilized as a basis for modifying or designing other embodimentsfor carrying out the same purposes of the present invention. Thoseskilled in the art will also appreciate that such equivalent embodimentsdo not depart from the spirit and scope of the invention as set forth inthe appended claims.

1. A peptide hybrid for protein immobilization composed of anoligopeptide comprising 7-17 amino acids having a specific proteinspecific affinity and PEG linked to the oligopeptide by covalent bond.2. The peptide hybrid for protein immobilization according to claim 1,wherein the protein is an antibody.
 3. The peptide hybrid for proteinimmobilization according to claim 1, wherein the oligopeptide iscomposed of 13-17 amino acids.
 4. The peptide hybrid for proteinimmobilization according to claim 1, wherein the oligopeptide containsthe amino acid sequence selected from the group consisting of sequencesrepresented by SEQ. ID. NO: 1-NO: 5 and formula
 4. 5. The peptide hybridfor protein immobilization according to claim 1, wherein the PEG (polyethylene glycol) has the molecular weight of 60-3000.
 6. The peptidehybrid for protein immobilization according to claim 1, wherein thehybrid is represented by Formulas 1-3.


7. The peptide hybrid for protein immobilization according to claim 6,wherein the hybrid is represented by formula
 1. 8. The peptide hybridfor protein immobilization according to claim 1, wherein the hybrid isrepresented by formula 1a. <Formula 1a> H₂N(CH₂CH₂O)₂CH₂CH₂COEDDC*AWHLGELVWC*T-CONH₂ (C*: disulfide bond;EDDC*AWHLGELVWC*T: SEQ. ID. NO: 4)


9. A method for preparing a protein monolayer comprising the followingsteps: 1) activating the carboxyl group included in a solid substrate;2) surface-treating the solid substrate of step 1) with the peptidehybrids for protein immobilization of claim 1; and, 3) binding a proteinonto the surface-treated solid substrate of step 2).
 10. The method forpreparing a protein monolayer according to claim 9, wherein the solidsubstrate is selected from the group consisting of CM-5 Au sensor chip,magnetic micro bead, glass plate, gold nano particle, biodegradableorganic polymer nano particle such as PLGA and various (micro)wellplates.
 11. A substrate for protein immobilization surface-treated withthe peptide hybrids for protein immobilization of claim
 1. 12. Animmunosensor having an antibody immobilized via the hybrid on solidsubstrate surface-treated with the hybrids for protein immobilization ofclaim
 6. 13. A detection method of an antigen comprising the step ofdetecting the antigen-antibody reaction after adding a sample antigen tothe antibody immobilized on the immunosensor of claim
 12. 14. Thedetection method of an antigen according to claim 13, wherein thedetection of the antigen-antibody reaction is performed by SPR, enzymeimmunoassay or fluorescence assay using a fluorescent probe.
 15. Thedetection method of an antigen according to claim 13, wherein thedetection is performed by the steps of binding an antigen specificantibody-coloring enzyme conjugate to the antigen; washing thereof;adding a substrate responding to the coloring enzyme; and measuring thecolor development.
 16. (canceled)
 17. The detection method of an antigenaccording to claim 13, wherein the detection is performed by the stepsof binding an antigen specific antibody-fluorescent probe conjugate tothe antigen; washing thereof; and measuring the fluorescence thereon.18. (canceled)
 19. A detection method of an antibody in a sample,comprising the following steps: 1) adding an antigen bindingspecifically to the antibody immobilized on the immunosensor and washingthereof; 2) adding a sample to the washed immunosensor of step 1) andwashing thereof; and, 3) detecting the antigen-antibody reaction betweenthe added antigen and the immobilized antibody.
 20. The detection methodaccording to claim 19, wherein the detection of the antigen-antibodyreaction of step 3) is performed by SPR, enzyme immunoassay orfluorescence assay using a fluorescent probe.
 21. The detection methodaccording to claim 19, wherein the step 3) is carried out as follows;binding the secondary antibody-coloring enzyme conjugate to the antibodyand washing thereof, and adding a substrate responding to the coloringenzyme and then measuring the color development.
 22. (canceled)
 23. Thedetection method according to claim 19, wherein the step 3 is carriedout by binding the secondary antibody-fluorescent probe conjugate to theantibody and washing thereof and measuring the fluorescence. 24.(canceled)